The widely popular class of quantum-dot molecular labels could so far not be utilized as standard fluorescent probes in STED (stimulated emission depletion) nanoscopy. This is because broad quantum-dot excitation spectra extend deeply into the spectral bands used for STED, thus compromising the transient fluorescence silencing required for attaining super-resolution. Here we report the discovery that STED nanoscopy of several red-emitting commercially available quantum dots is in fact successfully realized by the increasingly popular 775 nm STED laser light. A resolution of presently ∼50 nm is demonstrated for single quantum dots, and sub-diffraction resolution is further shown for imaging of quantum-dot-labelled vimentin filaments in fibroblasts. The high quantum-dot photostability enables repeated STED recordings with >1,000 frames. In addition, we have evidence that the tendency of quantum-dot labels to blink is largely suppressed by combined action of excitation and STED beams. Quantum-dot STED significantly expands the realm of application of STED nanoscopy, and, given the high stability of these probes, holds promise for extended time-lapse imaging.

Signal bias and membrane trafficking have recently emerged as important considerations in the therapeutic targeting of the glucagon-like peptide-1 receptor (GLP-1R) in type 2 diabetes and obesity. In the present study, we have evaluated a peptide series with varying sequence homology between native GLP-1 and exendin-4, the archetypal ligands on which approved GLP-1R agonists are based. We find notable differences in agonist-mediated cyclic AMP signalling, recruitment of β-arrestins, endocytosis and recycling, dependent both on the introduction of a His → Phe switch at position 1 and the specific mid-peptide helical regions and C-termini of the two agonists. These observations were linked to insulin secretion in a beta cell model and provide insights into how ligand factors influence GLP-1R function at the cellular level.

Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1R), glucose-dependent insulinotropic polypeptide (GIPR) and glucagon (GCGR) are important regulators of insulin secretion and energy metabolism. GLP-1R agonists have been successfully deployed for the treatment of type 2 diabetes, but it has been suggested that their efficacy is limited by target receptor desensitisation and downregulation due to recruitment of β-arrestins. Indeed, recently described GLP-1R agonists with reduced β-arrestin-2 recruitment have delivered promising results in preclinical and clinical studies. We therefore aimed to determine if the same phenomenon could apply to the closely related GIPR and GCGR. In HEK293 cells depleted of both β-arrestin isoforms the duration of G protein-dependent cAMP/PKA signalling was increased in response to the endogenous ligand for each receptor. Moreover, in wild-type cells, “biased” GLP-1, GCG and GIP analogues with selective reductions in β-arrestin-2 recruitment led to reduced receptor endocytosis and increased insulin secretion over a prolonged stimulation period, although the latter effect was only seen at high agonist concentrations. Biased GCG analogues increased the duration of cAMP signalling, but this did not lead to increased glucose output from hepatocytes. Our study provides a rationale for development of GLP-1R, GIPR and GCGR agonists with reduced β-arrestin recruitment, but further work is needed to maximally exploit this strategy for therapeutic purposes.

Abstract-We present a multi-color STED fluorescence microscope providing far-field optical resolution down to 20 nm for biomedical research. The optical design comprises fiber lasers, beam scanners, and a set of active and passive polarizing elements that cooperatively yield an optically robust system for routinely imaging samples at subdiffraction length scales.